tretinoin and 5-6-epoxyretinoic-acid

Topics: Animals; Kidney; Models, Biological; Tretinoin; Vitamin A

The nature of the products formed by reaction of all-trans retinoic acid (1) and its major metabolite, 5,6-epoxyretinoic acid (2), with the Fenton reagent was investigated. Oxidation of 1 in a vigorously stirred biphasic medium (0.1 M phosphate buffer, pH 7.4/ethyl acetate 5:1 v/v) with Fe2+/EDTA complex (2 mol equiv) and a 10-fold excess of H2O2 proceeded smoothly to give a very complex mixture of products. Repeated TLC fractionation of the reaction mixture after methylation allowed isolation of the main products which were identified as 2 methyl ester, (7E)-7,8-epoxyretinoic acid methyl ester (6), all-(E)-2,6-dimethyl-8-(2,6,6-trimethyl-2-cyclohexen-1-ylidene)-2,4,6-octatrienal (11), the novel (9E)-5,6,9,10-diepoxyretinoic acid methyl ester (7), and (9Z)-5,6,9,10-diepoxyretinoic acid methyl ester (8) (1:1 mixture of syn/anti isomers each), 5,6-epoxy-beta-ionone (9), 5,6-epoxy-beta-ionylideneacetaldehyde (10), and trace amounts of beta-ionone (12), beta-ionylideneacetaldehyde (13), and 4-oxoretinoic acid (3) methyl ester. When the oxidation was carried out with the substrate and the Fenton reagent at concentrations as low as 10 microM, the main detectable products were 2 methyl ester, 11, and 7/8. Under similar conditions, the epoxide 2 gave mainly products 7-10. A less efficient conversion of 1 and 2 but similar product patterns were observed with other oxidizing systems such as peroxidase/H2O2 and 13-hydroperoxyoctadecadienoic acid in the presence of Fe(II). Besides providing the first detailed insight into the products formed by reaction of a retinoid with the Fenton reagent, the results of this study disclose a novel nonenzymatic route from 1 to the epoxide 2 and offer an improved chemical basis to inquire into the mechanism of the antiinflammatory, antimutagenic, and cancer chemopreventive action of these retinoids.

Topics: Free Radicals; Hydrogen Peroxide; Iron; Oxidation-Reduction; Signal Transduction; Tretinoin

All trans retinoic acid (1), a cancer chemopreventive agent and a pluripotent morphogen, was found to react efficiently with nitrite ions in a biphasic system consisting of CH(2)Cl(2)/0.1 M phosphate buffer (pH 3) 1:1 v/v to give a complex mixture of nitration products. Repeated TLC fractionation of the reaction mixtures after methylation allowed isolation of the main products, which could be identified as the 12-nitro derivatives 3a,b and the decarboxylated 12,14-dinitro and 5,6-epoxy-14-nitro derivatives 4 and 5a by spectral analysis. Use of (15)NO(2)(-) followed by extensive 2D NMR analysis, including (1)H,(15)N heteronuclear multiple bond correlation experiments, allowed identification of nitronitrate derivatives as additional constituents of the mixture. Under similar conditions, 1 methyl ester gave mainly 3a,b. 5,6-Epoxyretinoic acid (2) reacted smoothly with acidic nitrite to give mainly 5a and its isomer 5b whereas its methyl ester afforded 14-nitro derivatives 9a,b as chief products. The observed patterns of reactivity along with mechanistic experiments would suggest that nitrite-induced nitration of 1 proceeds through complex reaction pathways set in motion by attack of NO(2) to the 12- and 14-positions. Separate experiments showed that 1 can inhibit nitrite-induced N-nitrosation of 2,3-diaminonaphthalene at pH values of 4 and 5.5, as well as decomposition of caffeic acid under similar conditions. Overall, these results provide the first detailed insight into the reaction behavior of a retinoid toward reactive nitrogen species and shed light on previously overlooked nitrite scavenging properties of 1 of potential relevance to the mechanism of its antiinflammatory, antimutagenic, and cancer chemopreventive action.

Topics: Antineoplastic Agents; Chromatography, High Pressure Liquid; Esters; Hydrogen-Ion Concentration; Nitrites; Reactive Nitrogen Species; Sodium Nitrite; Structure-Activity Relationship; Tretinoin

The metabolism of all-trans retinoic acid (ATRA) has been reported to be partly responsible for the in vivo resistance to ATRA seen in the treatment of human acute promyelocytic leukemia (APL). However, ATRA metabolism appears to be involved in the growth inhibition of several cancer cell lines in vitro. The purpose of this study was to evaluate the in vitro activity of the principal metabolites of ATRA [4-hydroxy-retinoic acid (4-OH-RA), 18-hydroxy-retinoic acid (18-OH-RA), 4-oxo-retinoic acid (4-oxo-RA), and 5,6-epoxy-retinoic acid (5,6-epoxy-RA)] in NB4, a human promyelocytic leukemia cell line that exhibits the APL diagnostic t(15;17) chromosomal translocation and expresses the PML-RAR alpha fusion protein. We established that the four ATRA metabolites were indeed formed by the NB4 cells in vitro. NB4 cell growth was inhibited (69-78% at 120 h) and cell cycle progression in the G1 phase (82-85% at 120 h) was blocked by ATRA and all of the metabolites at 1 microM concentration. ATRA and its metabolites could induce NB4 cells differentiation with similar activity, as evaluated by cell morphology, by the nitroblue tetrazolium reduction test (82-88% at 120 h) or by the expression of the maturation specific cell surface marker CD11c. In addition, nuclear body reorganization to macropunctated structures, as well as the degradation of PML-RAR alpha, was found to be similar for ATRA and all of its metabolites. Comparison of the relative potency of the retinoids using the nitroblue tetrazolium reduction test showed effective concentrations required to differentiate 50% of cells in 72 h as follows: ATRA, 15.8 +/- 1.7 nM; 4-oxo-RA, 38.3 +/- 1.3 nM; 18-OH-RA, 55.5 +/- 1.8 nM; 4-OH-RA, 79.8 +/- 1.8 nM; and 5,6-epoxy-RA, 99.5 +/- 1.5 nM. The ATRA metabolites were found to exert their differentiation effects via the RAR alpha nuclear receptors, because the RAR alpha-specific antagonist BMS614 blocked metabolite-induced CD11c expression in NB4 cells. These data demonstrate that the principal ATRA Phase 1 metabolites can elicit leukemia cell growth inhibition and differentiation in vitro through the RAR alpha signaling pathway, and they suggest that these metabolites may play a role in ATRA antileukemic activity in vivo.

Topics: Antineoplastic Agents; Cell Cycle; Cell Differentiation; Cell Division; Dose-Response Relationship, Drug; Granulocytes; Humans; Integrin alphaXbeta2; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Oncogene Proteins, Fusion; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Time Factors; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA), a signaling molecule derived from vitamin A, controls growth and differentiation of a variety of cell types through regulation of gene transcription. In the vertebrate retina, RA also regulates gap junction-mediated physiological coupling of retinal neurons through a nontranscriptional mechanism. Here we report that RA rapidly and specifically modulates synaptic transmission at electrical synapses of cultured retinal horizontal cells through an external RAR(beta)(/gamma)-like binding site, the action of which is independent of second messenger cascades. External application of all-trans retinoic acid (at-RA) reversibly reduced the amplitude of gap junctional conductance in a dose-dependent manner, but failed to affect non-gap-junctional channels, including glutamate receptors. In contrast, internal dialysis with at-RA was ineffective, indicating an external site of action. Selective RAR(beta)(/gamma) ligands, but not an RAR(alpha)-selective agonist, mimicked the action of at-RA, suggesting that gating of gap junctional channels is mediated through an RAR(beta)(/gamma)-like binding site. At-RA did not act on gap junctional conductance by lowering [pH](i) or by increasing [Ca(2+)](i). A G protein inhibitor and protein kinase inhibitors did not block at-RA uncoupling effects indicating no second messenger systems were involved. Direct action of at-RA on gap junction channels was further supported by its equivalent action on whole-cell hemi-gap-junctional currents and on cell-free excised patch hemichannel currents. At-RA significantly reduced single-channel open probability but did not change unitary conductance. Overall, the results indicate that RA modulates horizontal cell electrical synapses by activation of novel nonnuclear RAR(beta)(/gamma)-like sites either directly on, or intimately associated with, gap junction channels.

Topics: Adenosine Triphosphate; Animals; Bass; Cells, Cultured; Electrophysiology; Gap Junctions; Glucuronides; Guanosine Triphosphate; Receptors, Retinoic Acid; Retina; Retinoic Acid Receptor alpha; Synapses; Tretinoin

The effects of detergent, alamethicin (a channel-forming peptide), and the inducers phenobarbital and 3-methylcholanthrene on glucuronidation of all-trans-retinoic acid (atRA) and 5,6-epoxy-atRA have been investigated using liver microsomes from Sprague-Dawley and Fischer 344 rats. Conditions for enzymatic glucuronidation were optimized for substrate concentration, protein, and time by using atRA and Sprague-Dawley microsomes. With detergent-activated Sprague-Dawley microsomes, 5,6-epoxy-atRA was shown to be a significantly better substrate than atRA for microsomal glucuronidation (263 vs. 116 pmol/mg/min for 5,6-epoxy-atRA and atRA, respectively). The product of incubation of microsomes with atRA and UDP-glucuronic acid was identified as a glucuronide by beta-glucuronidase hydrolysis and by HPLC analysis. Alamethicin was shown to be a highly effective activator of glucuronidation activity; atRA and 5,6-epoxy-atRA glucuronidation rates were increased 2- and 3-fold, respectively, compared with detergent activation. Alamethicin (but not detergent) significantly increased retinoid glucuronidation by microsomes from Fischer 344 rats treated with phenobarbital and 3-methylcholanthrene, compared with untreated controls. The two compounds were equally effective inducers of activity, although 5,6-epoxy-atRA was again the better substrate. The same control and induced Fischer rat microsomes were photolabeled with [32P]5-azido-UDP-glucuronic acid in the absence or presence of detergent, two concentrations of alamethicin, and a 10-fold molar excess of unlabeled UDP-glucuronic acid. Photoincorporation into microsomal proteins from detergent-disrupted induced microsomes was 2-3 times greater than that of controls. Alamethicin increased photoincorporation of the probe into UDP-glucuronosyltransferase proteins an additional 1.5-2-fold in control and induced microsomes, compared with the respective detergent-activated samples.

Topics: Affinity Labels; Alamethicin; Animals; Enzyme Induction; Glucuronosyltransferase; Male; Methylcholanthrene; Microsomes, Liver; Phenobarbital; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; Tretinoin

The degradation products formed when 13-cis retinoic acid (13-cis RA) and all-trans RA were exposed to fluorescent light and air were investigated. These retinoids are known to undergo Z-E isomerization (due to the existence of four unsaturated double bonds) and oxidation when exposed to light and air. Analysis by LC was carried out on a 25 cm x 4.6 mm Zorbax Rx-SIL (5 microns) with a mobile phase (1.4 ml min-1) of heptane-THF-acetic acid (96.5:3.5:0.015) and an in-line UV (365 nm) detector. The LC eluate was coupled through a Vestec universal interface to a Finnigan 4023 mass spectrometer. EI-mass spectra were obtained at 77 eV from m/z 200 to 350 with multiplier voltage of 1200 V. Solid samples of 13-cis RA and all-trans RA exposed to light and air and also solutions of these retinoids in the mobile phase exposed to the same conditions were used for the analysis. Tentative identities of the degradation products from the mass spectra suggest the isomerization of the retinoids (Z-E isomerism) and the formation of the 5,6-epoxides of these isomers. Identities of the 5,6-epoxides were confirmed with chromatographic and mass spectral data from synthetic samples of the epoxides. Isomerization occurred more readily in solution than in the solid form and the 13-cis RA isomer oxidized more readily than the all-trans isomer.

Topics: Chromatography, Liquid; Fluorescence; Indicators and Reagents; Isomerism; Mass Spectrometry; Oxidation-Reduction; Spectrophotometry, Ultraviolet; Tretinoin

Topical all-trans retinoic acid (RA) modulates growth and differentiation of skin and is used in the treatment of various dermatological disorders. RA is metabolized to 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA, which are believed to be markedly less active than RA. 3,4-didehydroretinoic acid (ddRA) is a metabolite of 3,4-didehydroretinol which is present in skin. ddRA is biologically active and acts as a morphogen. We have determined the relative biological activity of ddRA, 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA as assessed by three retinoid responsive systems relevant to skin. RA, ddRA, 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA (10-100 nM) reduced epidermal transglutaminase activity in human keratinocytes to similar extents, and inhibited alpha-melanocyte-stimulating hormone-isobutylmethylxanthine-inducible tyrosinase activity in Cloudman S-91 mouse melanoma cells by 67, 39, 48, 51 and 19%, respectively, at 100 nM. Daily topical application of the retinoids to hairless mouse skin for 4 days resulted in dose-dependent changes in epidermal thickness and global histological score. The relative potencies of RA, ddRA, 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA, as calculated by parallel line assay, were 1.0, 0.60, 0.34, 0.29 and 0.18, respectively, for epidermal hyperplasia and 1.0, 0.78, 0.23, 0.14 and 0.08, respectively, for global histological score. Interestingly, the compounds exhibited a similar rank order of potency with respect to induction of cellular retinoic acid binding protein-II mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Animals; Carrier Proteins; Cells, Cultured; Epidermis; Humans; Keratinocytes; Male; Melanoma, Experimental; Mice; Mice, Hairless; Monophenol Monooxygenase; Receptors, Retinoic Acid; RNA, Messenger; Skin; Transglutaminases; Tretinoin; Tumor Cells, Cultured

Chlorogenic acid (3-O-caffeoylquinic acid) inhibited haematin- and haemoglobin-catalysed retinoic acid 5,6-epoxidation. Some other phenol compounds (caffeic acid and 4-hydroxy-3-methoxybenzoic acid) also showed inhibitory effects on the haematin- and haemoglobin-catalysed epoxidation, but salicylic acid did not. Of the above compounds, caffeic acid and chlorogenic acid were potent inhibitors compared with the other two, suggesting that the o-hydroquinone moiety of chlorogenic acid and caffeic acid is essential to the inhibition of the epoxidation. Although caffeic acid inhibited retinoic acid 5,6-epoxidation requiring the consumption of O2, formation of retinoic acid radicals was not inhibited on the addition of caffeic acid to the incubation mixture. The above results suggest that caffeic acid does not inhibit the formation of retinoic acid radicals but does inhibit the step of conversion of retinoic acid radical into the 5,6-epoxide.

Topics: Caffeic Acids; Chlorogenic Acid; Chromatography, High Pressure Liquid; Electron Spin Resonance Spectroscopy; Free Radicals; Heme; Hemin; Hemoglobins; Humans; Kinetics; Phenols; Tretinoin

Topics: Acetonitriles; Chromatography, High Pressure Liquid; Methanol; Retinaldehyde; Solvents; Tretinoin; Vitamin A

Retinoic acid 5,6-epoxidase activity was found in several hemoproteins such as human oxy- and methemoglobin (HbO2 and MetHb), equine skeletal muscle oxy- and metmyoglobin (MbO2 and MetMb), bovine liver catalase, and horseradish peroxidase. Hematin also catalyzed retinoic acid 5,6-epoxidation. The results suggest that the heme moiety participates in the epoxidation. However, neither horse heart cytochrome c, nor free ferrous ion nor free ferric ion exhibited the epoxidase activity. Some hemoproteins (HbO2, MetHb, MbO2, MetMb, catalase, peroxidase, and hematin) exhibited characteristic individual pH dependences of the activity, suggesting that the epoxidase activities of the hemoproteins are influenced by the apoenzymes to some degree. This view is also supported by the finding that preincubation of an HbO2 preparation at various temperatures (37-70 degrees C) reduced its epoxidase activity with increasing temperature, whereas the activity of hematin was unaffected. Active oxygen scavengers such as mannitol, catalase, and superoxide dismutase exhibited no effect on the epoxidase activities of HbO2, MetHb, MbO2, and MetMb. A ligand of heme, CN- (100 mM), inhibited the epoxidase activities but N3- (100 mM) did not. The epoxidase activities were completely inhibited by NADPH, NADH, and/or 2-mercaptoethanol but not by NADP+ and/or NAD+. An intermediate in the epoxidation may be reduced by NADPH, NADH and/or 2-mercaptoethanol. Radical species can be considered as plausible candidates for the intermediate.

Topics: Animals; Cattle; Chromatography, High Pressure Liquid; Hemeproteins; Horses; Hot Temperature; Humans; Hydrogen-Ion Concentration; Kinetics; Myoglobin; Oxidation-Reduction; Oxyhemoglobins; Spectrophotometry, Ultraviolet; Tretinoin

Rats on a normal diet were administered physiological doses of [3H]retinyl acetate or [3H]retinol orally for 5 days to label endogenous retinoid pools. The kidney retinoids were extracted and separated by DEAE-Sephadex into neutral and acidic fractions. All-trans-retinoic acid and 5,6-epoxyretinoic acid were isolated and unequivocally identified by chromatographic analysis, chemical derivatization, and mass spectroscopy. The identities of retinol and retinyl palmitate were verified by high performance liquid chromatography and reactivity with trifluoroacetic acid. Control experiments showed that retinoid epoxidation truly occurred in vivo. The specific radioactivities of the recovered acidic retinol metabolites were similar to those of the recovered neutral retinoids. Thus, retinoic acid and its metabolite 5,6-epoxyretinoic acid are endogenous rat kidney retinoids which are in the pathway of retinol metabolism under physiological conditions. The concentrations of retinyl palmitate (8.7 microM), retinol (4.6 microM), all trans-retinoic acid (1.3 microM) and 5,6-epoxyretinoic acid (0.25 microM) measured indicate that acidic retinoids are comparatively significant vitamin A metabolites in kidney.

Topics: Animals; Chromatography, High Pressure Liquid; Diterpenes; Kidney; Male; Mass Spectrometry; Rats; Rats, Inbred Strains; Retinyl Esters; Tretinoin; Vitamin A

The major metabolite in the small intestinal mucosa of vitamin A deficient rats dosed intrajugularly with 5,6-epoxy[3H]-retinoic acid has been identified as 5,6-epoxyretinoyl beta-glucuronide. The assignment was based on the metabolite's chemical, spectral, and chromatographic properties. Incubation of the metabolite with beta-glucuronidase released 5,6-epoxyretinoic acid. Incubation of 5,6-epoxyretinoic acid with rat liver microsomes in the presence of uridine-5'-diphospho-1 alpha-D-glucuronic acid produced the metabolite. 5,6-Epoxy[3H]retinoyl beta-glucuronide weas observed in the liver, small intestinal mucosa, and intestinal contents but not in kidney of vitamin a deficient rats. Its concentration was greatly diminished in liver and small intestinal mucosa, and it was not observed in kidney of vitamin A deficient rats dosed orally with retinoic acid for several days before administration of 5,6-epoxy[3H]retinoic acid. Generally, oral retinoic acid treatment accelerated 5,6-epoxyretinoic acid metabolism and enhanced accumulation of highly polar metabolites. Moreover, 5,6-epoxyretinoic acid metabolism was more rapid than that of retinoic acid and did not result in production of retinoic acid.

Topics: Animals; Chromatography, High Pressure Liquid; Intestinal Mucosa; Intestine, Small; Male; Microsomes, Liver; Rats; Rats, Inbred Strains; Tretinoin; Vitamin A Deficiency

A method is described for measuring the relative activities of vitamin A active substances based on their direct effect on the cornified vaginal epithelium of vitamin A-deficient rats. The results obtained agree well with those found in the tracheal organ culture assay. The relative activities found for several test compounds were: all-trans-retinoyl-beta-glucuronide greater than all-trans-retinoic acid greater than all-trans retinol greater than all-trans-5,6-epoxyretinoic acid. The assay is simple and inexpensive to perform, and should find use in laboratories where the equipment and personnel required for the tracheal organ culture assay are not available.

Topics: Animals; Biological Assay; Castration; Dose-Response Relationship, Drug; Epithelium; Female; Glucuronates; Rats; Tretinoin; Vaginal Diseases; Vaginal Smears; Vitamin A; Vitamin A Deficiency

A description of the enzyme that produces 5,6-epoxyretinoic acid from all-trans-retinoic acid has been presented. This enzyme system is found in highest concentrations in the kidney followed by intestine, liver and spleen. The enzyme requires molecular oxygen, magnesium ions, ATP, and NADPH. In the kidney, it is found in the mitochondrial and microsomal fractions and has a Michaelis constant of 3.2 X 10(-6) M and 3.7 X 10(-6) M for 13-cis and all-trans-retinoic acid, respectively. The resultant product, 5,6-epoxyretinoic acid, has minimal activity in supporting growth of vitamin A-deficient rats, its activity estimated to be 0.5% that of retinoic acid. An investigation of the biliary excretion products of tritiated retinoic acid has revealed several unknown metabolites. A glucuronidase sensitive metabolite from these products has been isolated and identified as retinoyl-beta-glucuronide by ultraviolet absorption spectrometry and mass spectrometry. The retinoyl-beta-glucuronide originally discovered by Olson and collaborators accounts for only 12% of the total excreted biliary products of retinoic acid. At least four to six major unknown retinoic acid metabolites, in addition to retinoyl-beta-glucuronide, have been detected and will shortly be identified.

Topics: Animals; Bile; Chromatography, High Pressure Liquid; Epoxy Compounds; Glucuronates; Kidney; Rats; Tretinoin; Vitamin A Deficiency

5,6-Epoxyretinoic acid was detected in small intestine, kidney, liver, testes and serum of vitamin A-deficient rats 3 h after a single physiological dose of [3H]retinoic acid. The maximum concentration of 5,6-epoxide in intestinal mucosa was observed 3 h after intrajugular administration of retinoic acid. However, at 7 h post administration, no 5,6-epoxyretinoic acid was detected in mucosa, demonstrating the rapid intestinal metabolism or excretion of this metabolite. No 5,6-epoxy[3H]retinoic acid was detected in mucosa, liver or serum of retinoic acid-repleted rats 3 h after administration of 2 micrograms of [3H]retinoic acid.

Topics: Animals; Chromatography, High Pressure Liquid; Epoxy Compounds; Kinetics; Male; Rats; Tissue Distribution; Tretinoin; Vitamin A Deficiency

The potential of all-trans-5,6-epoxyretinoic acid to promote growth in vitamin A-deficient rats was evaluated over a 25-fold dose range using single daily intraperitoneal injections. 5,6-Epoxyretinoic acid was found to be 0.5% as active as all-trans-retinoic acid. The discrepancies between the present results and those obtained previously by other workers are discussed.

Topics: Administration, Oral; Animals; Dose-Response Relationship, Drug; Epoxy Compounds; Growth; Injections, Subcutaneous; Male; Rats; Tretinoin; Vitamin A Deficiency

Topics: Animals; Biotransformation; Cattle; Choline; Epoxy Compounds; Lymphocytes; Membrane Lipids; Phorbols; Phospholipids; Structure-Activity Relationship; Tetradecanoylphorbol Acetate; Tretinoin